Potential pharmaceuticals targeting neuroimmune interactions in treating acute lung injury

Abstract Background and main body Although interactions between the nervous and immune systems have been recognized decades ago, it has become increasingly appreciated that neuroimmune crosstalk is among the driving factors of multiple pulmonary inflammatory diseases including acute lung injury (ALI). Here, we review the current understanding of nerve innervations towards the lung and summarize how the neural regulation of immunity and inflammation participates in the onset and progression of several lung diseases, especially ALI. We then present advancements in the development of potential drugs for ALI targeting neuroimmune interactions, including cholinergic anti‐inflammatory pathway, sympathetic‐immune pathway, purinergic signalling, neuropeptides and renin‐angiotensin system at different stages from preclinical investigation to clinical trials, including the traditional Chinese medicine. Conclusion This review highlights the importance of considering the therapeutic strategy of inflammatory diseases within a conceptual framework that integrates classical inflammatory cascade and neuroimmune circuits, so as to deepen the understanding of immune modulation and develop more sophisticated approaches to treat lung diseases represented by ALI. Key points The lungs present abundant nerve innervations. Neuroimmune interactions exert a modulatory effect in the onset and progression of lung inflammatory diseases, especially acute lung injury. The advancements of potential drugs for ALI targeting neuroimmune crosstalk at different stages from preclinical investigation to clinical trials are elaborated. Point out the direction for the development of neuroimmune pharmacology in the future.

• The advancements of potential drugs for ALI targeting neuroimmune crosstalk at different stages from preclinical investigation to clinical trials are elaborated.
• Point out the direction for the development of neuroimmune pharmacology in the future.

INTRODUCTION
Acute respiratory distress syndrome (ARDS) is thought to be a severe form of acute lung injury (ALI), which has an acute onset and rapid progression and is often characterized by diffuse lung inflammation and oedema. 1 ALI or ARDS is caused by multiple predisposing conditions, which can be roughly divided into sepsis and non-sepsis etiologies. 2 The limited means of prevention and treatment contribute to high mortality and morbidity of the disorder.
In the LUNG SAFE study 3 concerning patients admitted to ICUs of 50 countries, 10.4% of the patients met ARDS criteria with hospital mortality rates of 34.9%, 40.3% and 46.1% for mild, moderate and severe ARDS, respectively.Given the geoeconomic variation, PRoVENT-iMiC 4 (international observational research of ICU patients from 10 Asian middle-income countries) exhibited a lower incidence (7%) but higher mortality rate (45%) of ARDS compared to the LUNG SAFE study.Besides, another multicenter cohort study on patients surviving 2 years after ALI indicated a median cost of $35 259 for every survivor, which brought a significant economic burden to both the families and the healthcare system. 5][8][9] Despite great efforts made in developing novel pharmacotherapies for ALI, 10 very few have shown clinical efficacy, which has troubled physicians for a long time.
Actually, of all the potential interventions, only low tidal volume ventilation has been demonstrated to be beneficial for the patients. 11Therefore, discovering new pathogenesis and related therapeutic approaches to manage ALI is of great importance to a better clinical prognosis.
Recently, researchers have paid more attention to neuroimmune interactions, which serve as an underestimated novel mechanism in regulating excessive inflammatory responses of various disorders.The nerve system is capable of receiving stimuli and relaying signals in the form of neurotransmitters and/or neuropeptides to the adja-cent immune cells via specific receptors, the cells can also release mediators, which in turn stimulate nearby endings of peripheral nerve. 12These close interactions between neurons and immune cells have been found to be a key mechanism driving the genesis of multiple inflammatory diseases. 13,14In the development of ALI, immune cells are also innervated by neural activity, 15,16 and manipulation of neurotransmitter/neuropeptide or their receptors may become a new strategy to treat the syndrome.Accordingly, multiple agents have emerged to potently regulate the neuroimmune crosstalk, aiming to inhibit the diseaseassociated inflammatory overactivation.
In this review, we first summarize the anatomic nerve innervations towards the lung, mainly including afferent innervation, central nervous system (CNS), and efferent innervation (Figure 1).We then recapitulate how the neural regulation of immunity and inflammation participates in the onset and progression of several pulmonary diseases, especially ALI (Figures 2-4).In addition, we focus on the current knowledge of potential drugs targeting the cholinergic anti-inflammatory pathway (CAP), sympatheticimmune pathway, purinergic signalling, neuropeptides, and renin-angiotensin system (RAS) at different stages from preclinical investigation to clinical trials, including the traditional Chinese medicine, as well as discuss their promising usages in treating ALI/ARDS (Figure 5 and Tables 1-5).This review deepens the understanding of immune modulation and develops more sophisticated approaches to treat lung diseases represented by ALI.

NERVE INNERVATION TOWARDS THE LUNG
The nerve system is categorized as CNS including the spinal cord and brain, and the peripheral nervous system, which dominates the lung and is broadly divided into the afferent sensory nerve system and the efferent nerve system.Within the latter, the autonomic nerve is located in the viscus and controls smooth muscles, cardiac muscles, and glands.It can be further classified into parasympathetic and sympathetic nerves according to their functional features (Figure 1).

F I G U R E 1
Overview of innervation in the lung.The lung is mainly innervated by sensory neurons arising from the jugular nodose complex or dorsal root ganglia (shown in blue), parasympathetic neurons originating from the brainstem or vagus nerve (shown in orange), and sympathetic neurons coming from the thoracic ganglia (shown in red).nAChR, nicotinic acetylcholine receptor; 5-HT, 5hydroxytryptamine; TRPV/A1, Transient receptor potential vanilloid 1/ ankyrin 1; GPCR, G-protein-coupled receptor; AT1R, angiotensin II type 1 receptor; AT2R, angiotensin II type 2 receptor; ACh, acetylcholine; ASM, airway smooth muscle.

Afferent innervation
The afferent nerves of the lung and respiratory tract are mainly the afferent fibers of the vagus nerve (about 20%), which communicate the external and internal environments to the CNS.Most of the afferent nerve cell bodies are located in nodose or jugular vagal ganglia, while a small proportion of afferent nerves come from the thoracic dorsal root ganglia.Vagal sensory fibers are classified into fastest Aβ-fibers, intermediate Aδ-fibers and slowest unmyelinated C-fibers according to their conduction velocities.A-fibers are often activated by mechanical forces such as stretch and touch, but they are not sensitive to chemical stimuli.On the contrary, C-fibers can be activated by chemical, thermal and physical stimuli.In addition, Aβ-fiber mechanoreceptors are subcategorized as rapidly and slowly adapting receptors based on the action poten-tial adaptation.Any circumstances altering the balance of mechanical forces, such as bronchoconstriction resulting from oedema or mucus overproduction, can lead to the activation of the two receptors. 17A long list of chemicals causes a mechanical action on airway structural cells, followed by indirect sensitization of the receptors.Yet, adenosine triphosphate (ATP) itself can directly activate the intrapulmonary mechanoreceptors through P2X2/3 purinergic receptors on most nodose neurons. 18,19nmyelinated C-fibers outnumber A-fibers at a ratio of around 8:1 in the afferent nerves of the airway, 20 and are subclassified as nodose and jugular C-fibers according to their locations.Nodose C-fibers are responsive to adenosine, ATP and 5-hydrotryptamine, whereas jugular C-fibers express substance P and calcitonin gene-related product (CGRP), which directly induce the contraction of airway smooth muscle (ASM). 21Besides, C-fibers express a spectrum of receptors, mainly including ligand/voltagegated ion channels and G-protein-coupled receptors (GPCRs).Among ionotropic receptors, the well-studied nicotinic cholinergic receptor (nAChR) is a pentamer made up of 17 subunits α1-10, β1-4, γ, δ and ε, spanning the membrane four times. 22Interestingly, this type of receptor is unlikely to be stimulated by endogenous acetylcholine (ACh) from postganglionic cholinergic nerves but is responsive to epithelial-derived ACh. 23As for the 5hydrotryptamine receptor family, all members belong to GPCRs except for the 5-hydrotryptamine-3 receptor, which is an ionotropic receptor that is selectively expressed in nodose sensory neurons, 24 and its activation leads to C-fibers action potential discharge. 25,26Purine receptors are divided into P1 (adenosine-activated) and P2 (ATP or adenosine diphosphate-activated) types. 279][30][31] P2 receptors include two main families, namely, P2X (ligand-gated ion channels) with seven subtypes (P2X1-7) and P2Y (GPCRs) also with 7 subtypes (P2Y1, 2, 4, 6 and 11-14). 32TP depolarizes all nodose neurons via the heteromeric channel P2X2/P2X3, but not in C-fibers of jugular neurons, on which P2X2 expression is absent. 19,33

Central nervous system
The brainstem is the destination of all afferent sensory nerves within vagus nerves, and also the place where afferent nerves innervate the second-order neurons, ascending to the higher brain region, descending to the spinal cord and projecting to adjacent brain stem nuclei.The embryological difference between nodose and jugular C-fibers is also reflected in the destinations with the former tending to the medullary region in the nucleus of the solitary tract and adjacent postrema area, while the latter favouring the trigeminal nucleus area. 34,35C-fibers and most A-fibers terminating in the nucleus of the solitary tract depend on non-N-methyl-D-aspartate receptors to complete the  glutamatergic transmission, whereas a small portion of Aδ-fibers requires N-methyl-D-aspartate receptors. 36However, current knowledge about neurons in the solitary tract area remains limited, let alone neurons in the trigeminal nucleus area.Kinin receptors B1 and B2 belong to the GPCR family and are expressed in CNS 37 ; besides, the B1 receptor is normally absent but rapidly and highly upregulated under inflammatory circumstances. 38Another member of GPCRs located in the brain is the opioid receptors, which are mainly classified into three classic types: µ, δ and κ.The intriguing receptors can be activated by both endogenous opioid peptides and exogenous opiate compounds. 39A B L E 3 The development status of drugs targeting purinergic signalling.

Drugs Utility
Preclinical The RAS comprises the angiotensin I-converting enzyme (ACE)-angiotensin (Ang) II-angiotensin receptor (ATR) axis and the ACE2-Ang (1-7)-Mas receptor axis and is well known for its bidirectional role in the cardiovascular and urinary systems.All Ang peptides are derived from the cleavage of angiotensinogen in CNS, with the majority being produced by astrocytes and constitutively secreted into the interstitial space and cerebrospinal fluid. 40The angiotensinogen is then decomposed into Ang I by aspartyl-protease rennin and further turned into active Ang II by ACE. 41The function of Ang II depends on its binding to distinct receptors, AT1R and AT2R, which are seven transmembrane GPCRs. 42pecifically, the two receptors mediate opposite effects: AT1R plays a significant role in inflammation, fibrosis, proliferation and vasoconstriction, 43 while AT2R promotes anti-inflammation, antifibrosis and vasodilation. 44dditionally, ACE2 is a homolog of ACE and cleaves Ang II into Ang (1-7), which exerts lung protective actions via its Mas receptor.

Parasympathetic nerve
Most of the cholinergic parasympathetic nerves controlling tracheal walls arise from the ambiguous nucleus of the medulla and travel within the vagus nerves to intramural ganglia within airways, 46 while a smaller part comes from the dorsal motor nucleus of the vagus nerves. 47After arriving at the intramural ganglia, parasympathetic nerves project to postganglionic neurons and send information to airway effector cells by releasing ACh. 48

Sympathetic nerve
The primary cell bodies of preganglionic sympathetic neurons are located in the upper six thoracic segments of the spinal cord, followed by projecting to secondary neurons in paravertebral sympathetic chain ganglia.The postganglionic sympathetic neurons originating from stellate and sympathetic chain ganglia dominate the lung, while those derived from stellate and superior cervical ganglia innervate the trachea. 49The termination of sympathetic nerve fibres is close to blood vessels, submucosal glands and ASM, 50,51 whose typical catecholamine neuromediators include noradrenaline and dopamine.

Pulmonary infection
Neuroimmune interactions regulate innate immune defence within the lung, and whether the effect is beneficial or harmful depends on distinct physiological and pathological situations.In Staphylococcus aureus pneumonia, transient receptor potential vanilloid 1 (TRPV1)-expressing sensory neurons suppress neutrophils infiltration into the lung and γδT cell effector functions, revealing a weakened antibacterial immunity.Otherwise, ablating the neurons or blocking CGRP can improve the overall survival of mice against lethal S. aureus infection. 52he expressions of TRPV1 and transient receptor potential ankyrin 1 in sensory neurons are enhanced shortly after rhinovirus infection, accompanied by elevated cough reflex sensitivity. 53In contrast, α7nAChR activation on bone-marrow-derived macrophage alleviates lung inflammation in influenza-exposed mice. 54Vagal sen- sory neurons within airways also respond to influenza and express antiviral or pro-inflammatory genes, which is accompanied by an increased leukocyte migration into the vagal ganglia. 55As for efferent innervation, the influenza virus activates the sympathetic nerves in the mouse lung, and peripheral sympathectomy improves the survival rate and restrains excess inflammation in the mice. 56Furthermore, the RAS plays a controversial role in the pathogenesis of ALI triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which recognizes ACE2 on the cell surface via a spike binding domain, followed by membrane fusion and virus uptake. 57Experimental data indicate that the increased ACE2 expression by RAS blockers may facilitate viral cell entry. 58Conversely, nuclear ACE2 inhibitor reduces viral replication and pulmonary inflammation in SARS-CoV-2-infected hamsters. 59On the other hand, ACE2 is a critical counter-regulator of the RAS and may exert lung protective effects in COVID-19.The above findings emphasize the necessity of solid evidence-based data in future research.Besides, the oestrogen-protected endothelial integrity through activating Ang (1-7)/Mas receptor signalling probably explains the sex differences in mobility and mortality during the SARS-CoV-2 attack. 60Figure 2)

Bronchial asthma
Bronchial asthma (or asthma) is orchestrated by various inflammatory cells and airway structural cells, including innate lymphoid cell (ILC)2, eosinophil, dendritic cells, as well as epithelial cells and ASM cells.Increasing evidence has linked the nervous system to inflammatory responses in asthma.Sensory neurons expressing TRPV1 and/or voltage-gated sodium channel Nav1.8 produce neuropeptides such as CGRP, vasoactive intestinal peptide (VIP) and substance P, which participate in the formation of asthma. 61CGRP is a negative regulator of ILC2-mediated allergic inflammation via its receptor subunits Calcrl and Ramp1. 62CGRP suppresses interleukin (IL)-33-induced ILC2 proliferation and subsequent eosinophil recruitment into the lung, thus protecting against tissue damage in allergic asthma models. 63CGRP also inhibits the maturation and function of dendritic cells, as well as regulates adaptive immune response in asthma. 64In ovalbumin or house dust mite-challenged asthmatic mice, IL-5 overproduction by the activated immune cells acts on TRPV1 and Nav1.8 to secret VIP, which stimulates resident ILC2s and T helper 2 cells, creating a positive feedback loop that aggravates allergic lung inflammation. 65,66Additionally, asthma patients and animal models exhibit enhanced cholinergic innervation compared with healthy individuals.Mechanistically, ACh is known to induce mucus hypersecretion and bronchoconstriction through muscarinic ACh receptor (M) 3 on airway epithelial cells and ASM cells, while the M 2 receptor provides negative feedback on ACh secretion. 67As for the sympathetic nerve system, tyrosine hydroxylase-expressing neurons activate β 2 adrenergic receptor (AR) by noradrenaline secretion to restrain ILC2-mediated inflammation. 68Besides, the postnatal transformation of the dopaminergic nerve to the adrenergic nerve is reported to be associated with asthma susceptibility in childhood through dopamine receptor D4 69 (Figure 3).

Chronic pulmonary obstructive disease
As another common chronic respiratory disease, chronic pulmonary obstructive disease (COPD) is a major worldwide health problem characterized by irreversible airflow limitation and abnormal inflammatory response in the airway.Overactivated ATP signalling through P2X2/3/4 receptors in sensory neurons is observed in COPD animal models and related to pro-inflammatory mediators generation, cough and bronchoconstriction. [70][71][72] Comparatively, α7nAChR activation in immune cells exhibits modulatory actions in COPD by limiting the production of inflammatory markers linked to disease severity. 73The role of VIP in COPD is controversial, to be specific, the elevated serum VIP level has been identified to be associated with COPD exacerbation, but inhaled VIP is beneficial for life quality in COPD patients. 74,75Besides, researchers also report the increased sputum CGRP and substance P levels in COPD patients, but the underlying mechanisms are largely obscure. 76,77Parasympathetic release of ACh via vagus nerves has been well known to activate M 3 muscarinic receptor, thus inducing bronchoconstriction, 78 whereas M 3 antagonist serves as a regular bronchodilator drug for COPD patients. 79Increased muscle sympathetic nerve activity caused by long-term hypoxia is another characteristic of COPD and often accompanied by systemic inflammation, with the mechanisms unexplored. 80nterestingly, despite the attenuation of hyperinflation, the long-acting inhaled β-agonist does not appear to affect sympathoexcitation 81 (Figure 3).

Acute lung injury
The pulmonary nervous system also plays a critical role in modulating acute inflammation of ALI.Pro-inflammatory macrophage-derived cytokine storms in ALI can be read by sensory neurons within vagus nerves and the signals are sent to the brain 82 ; subsequently, efferent parasympathetic neurons release ACh to stimulate α7nAChR on post-ganglionic neurons. 83The efferent fibres of vagus nerves are connected to the splenic nerves in the abdominal mesenteric ganglia, transmitting anti-inflammatory signals to the spleen.The splenic nerve endings release noradrenaline, which then stimulates specific T lymphocytes expressing choline acetyltransferase to synthesize ACh.Thereafter, ACh inhibits inflammatory factor production by activating the α7nAChR in immune cells. 84his process is known as the CAP.Commonly, stimulation of vagus or splenic nerves exerts an anti-inflammatory effect on lipopolysaccharide (LPS)-induced immune cells; while vagotomy aggravates the inflammation. 83In the ventilator-induced ALI rat model, pharmacological or electrical stimulation of vagus nerves ameliorates pulmonary injury via the α7nAChR-dependent pathway. 85imilarly, α7nAChR agonist can attenuate hyperoxiainduced ALI via alleviating the accumulation of high mobility group box 1 protein (HMGB1) in airways and circulation. 16][91][92] Besides, some studies have identified the protective roles of exogenous neuropeptides VIP and CGRP in improving lung damage in LPS-induced ALI, 93-95 while others have illustrated that CGRP antagonist decreases vascular permeability caused by LPS. 96,97This may be attributed to the complex pathological process of ALI, and whether one strategy takes precedence over the other may quite depend on the specific situation in the disorder (Figure 4).

Therapeutic agents targeting the CAP
The CAP plays a crucial role in regulating inflammation in ALI/ARDS.The LPS-induced inflammatory markers of ALI show significantly higher levels in cholinergicdeficient than wild-type mice, while selective α7nAChR agonist PNU282987 can decrease neutrophil accumulation, IL-1β and chemokine (C-X-C motif) ligand-1 levels in the lung of ALI mice. 98As another agonist towards α7nAChR, GTS-21 reduces neutrophilic inflammation in renal ischemia-reperfusion-triggered ALI mice; splenectomy or splenic macrophage depletion limits its protective effect, indicating engagement of these cells in CAP. 99n the mechanical ventilator-challenged mouse model, GTS-21 attenuates the lung and plasma levels of tumour necrosis factor (TNF)-α, as well as lung injury.However, modulating endogenous cholinergic signalling by mecamylamine and neostigmine can not affect the inflammatory response, implying that selective CAP stimulation may offer a new therapeutic strategy for this type of ALI. 100 PHA568487 and nicotine, the selective and nonselec-tive α7nAChR agonists, both alleviate pulmonary damage in gram-negative sepsis or severe acute pancreatitisassociated ALI. 101,102HMGB1 serves as a pivotal upstream regulator of inflammatory responses and correlate significantly with the severity in multiple diseases like ALI.A central acetylcholinesterase inhibitor Galantamine, as well as GTS-21, are able to protect against ALI in animal models by attenuating HMGB1 accumulation in lung tissues. 103,104Recently, Zhang et al. comprehensively investigated the protective role of the CAP pathway in the ALI model: the nAChR agonists monepantel and lobeline, the specific α7nAChR agonists AR-R17779 hydrochloride and GTS-21, as well as the anticholinesterase neostigmine attenuated LPS-induced pulmonary inflammation and tissue injury in ALI mice.Moreover, the authors conducted a pilot, nonrandomized, open-label and controlled clinical trial of lobeline in the management of ALI/ARDS, which exhibited higher ventilator-free days and ICU survival rate, along with decreased levels of inflammatory biomarkers in bronchoalveolar lavage fluid (BALF) and blood samples from patients within the lobeline group. 105Thus, nAChR agonist appears to be a potential therapeutic target for ALI.
Muscarinic M 3 receptor has been shown to participate in LPS-mediated pulmonary microvascular endothelial injury, which could be improved by the anticholinergic penehyclidine hydrochloride through the blockage of the M 3 receptor. 106,107Besides, non-selective muscarinic cholinergic receptor antagonist atropine or selective M 3 antagonist 4-DAMP, rather than M 1 or M 2 antagonist, shows regulatory effects on neutrophil infiltration, microvascular permeability and cytokines secretion in the lung of ALI mice, indicating the important role of M 3 receptor in LPS-stimulated pulmonary inflammation 108 (Figure 5A and Table 1).

Potential drugs targeting sympathetic-immune pathway
Dexmedetomidine, a highly selective α 2 AR agonist with analgesic and sedative effects, is one of the most wellknown drugs for clinical therapy of ARDS.0][111][112][113][114][115][116] BRL44408 maleate is a specific α 2A AR antagonist and has been validated to improve sepsis-associated ALI by suppressing proinflammatory mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways, 89 as well as by downregulating the HMGB1 level. 117Esmolol is a selective β 1 AR blocker applied for various types of tachycardia.
Interestingly, it also abates the increases in TNF-α, IL-6 and myeloperoxidase activity in BALF samples or lung tissues, thereby reducing disease severity and improving survival time in acute pancreatitis-related ALI rats. 90Similarly, landiolol hydrochloride also exhibits therapeutic effects on the LPS-challenged ALI model by decreasing pulmonary TNF-α, IL-6 and endothelin-1. 91he β 2 AR agonists are the most widely prescribed medication for chronic airway diseases like COPD and asthma.Recently, they have also been extensively investigated in experimental and clinical ALI.Among them, albuterol and formoterol confine excess lung inflammation by inhibiting the c-Jun N-terminal kinase/MAPK pathway, while terbutaline improves alveolar-capillary barrier function and enhances alveolar fluid clearance in ALI animal models. 92,118To determine whether an β 2 AR agonist would improve clinical outcomes in patients with ALI, a multi-centre, phase III randomized, placebocontrolled clinical trial was conducted among the patients receiving aerosolized albuterol or saline every 4 h for up to 10 days.However, by measuring the ventilator-free days as a primary outcome variable, the researchers concluded that routine use of β 2 AR agonists did not improve the prognosis in mechanically ventilated ALI patients (NCT 00434993). 119,120In another randomized placebocontrolled trial conducted at 12 UK centres, patients undergoing elective esophagectomy were randomly allocated to inhaled salmeterol or matched placebo treatment.The results showed that salmeterol administration did not prevent early ALI or improve organ failure, survival, or health-related quality of life, though it did downregulate biomarkers of airway inflammation and epithelial injury in these patients. 121,122n the peripheral dopaminergic nervous system, SKF38393 is a selective agonist for dopamine D1/D5 receptors.Recently, we found that SKF38393 administration inhibited excessive inflammation and oxidative stress in macrophages, as well as maintained airway epithelial barrier function in LPS-stimulated ALI mice, partly via the activation of nuclear factor erythroid-2-related factor 2 antioxidative system. 123Another D1 receptor agonist A68930 is also demonstrated to exhibit therapeutic efficacy on spinal cord injury-induced ALI rats by dampening the activation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) signaling. 124In addition, pretreatment with dopamine or D2 receptor-specific agonist quinpirole significantly increases survival rates and reduces neutrophil recruitment and pulmonary edema of LPS-challenged mice.However, the effects are not observed in D2 receptor knockout mice, suggesting its role in dopamine-mediated barrier protection. 125onversely, as a dopaminergic antagonist, domperidone aggravates LPS-induced TNF-α and IL-6 production in BALF of ALI mice, which may exacerbate the subsequent inflammatory injury 126 (Figure 5B and Table 2).

Pharmacological strategies targeting purinergic signalling
Transfusion-related ALI is a critical post-transfusion respiratory syndrome, but the underlying biological responses remain unclarified.The ATP-gated P2X1 cation channel is reported to participate in the development of the syndrome, with its selective antagonist NF449 improving overall survival, reducing BALF protein leakage and lung interstitial oedema in the mouse model by targeting monocytes/macrophages. 87Likewise, the P2X7 blockers A438079 suppresses LPS-stimulated pro-inflammatory cytokines release, neutrophil accumulation, and cysteinyl aspartate specific proteinase 1 activation by NLRP3 inflammasome in the ALI mouse model. 88Physalin D is extracted from Physalis angulata L. leaves and is capable of reversing ATP/LPS-induced lung injury in mice through inhibiting P2X7 receptor function. 127Increasing studies have indicated an essential role of platelets in ALI pathogenesis; P2Y12 antagonist clopidogrel treatment or P2Y12 knockout diminishes platelet activation, platelet-leukocyte aggregates and subsequent lung damage in mice induced by sepsis. 128Consistently, P2Y12 inhibitors clopidogrel and ticagrelor are also able to reduce the inflammatory response, as well as improve the migration, function and permeability of endothelial cells in the LPS-challenged cell model. 86enerally, activation of the adenosine A2A receptor is considered to be anti-inflammatory.As an A2A receptor agonist, polydeoxyribonucleotide downregulates the production of LPS-elevated pro-inflammatory cytokines and apoptotic factors, and promotes the recovery of injured lung of ALI rats via potently attenuating MAPK/NF-κB signaling pathway; meanwhile, the A2A receptor blocker 7-dimethyl-1-propargylxanthine reverses the effects. 129CGS21680 hydrochloride is also an A2A receptor-specific agonist and is confirmed to abrogate macrophage activation and histologic lung injury in LPS plus high oxygen-exposed mice. 130Cannabidiol is a non-psychotropic plant-derived cannabinoid with potent immunomodulatory properties.Cannabidiol administration decreased leukocyte migration, alveolar protein leakage, pro-inflammatory cytokines generation, and tissue damage in the LPS-stimulated ALI mouse model.The underlying mechanism may be associated with the activation of the A2A receptor since a selective A2A antagonist ZM241385 can abrogate the protective effect of Cannabidiol. 131In addition, peroxisome proliferatoractivated receptor-γ has been reported to upregulate A2A receptor expression in the ALI mouse lungs via binding to a DR10 response element within its premotor region.Hence, the combination of peroxisome proliferator-activated receptor-γ and A2A agonists is found to be a more efficient therapeutic strategy for ALI. 132Nevertheless, in a neurogenic ALI mouse model caused by severe traumatic brain injury (TBI), A2A receptor agonist CGS21680 exacerbates, whereas the inhibitor ZM241385 mitigates the inflammatory damage within lung tissues, which is attributed to the elevated plasma glutamate after severe TBI. 133,134The results indicate that when targeting the A2A receptor, full consideration should be given to whether it is nonneurogenic or neurogenic ALI; besides, combined therapy targeting both A2A and blood glutamate might be a potential strategy for TBI-ALI management.
The adenosine A2B receptor may also be a potential therapeutic target for ALI/ARDS.In an ALI model caused by LPS, simvastatin protects rats from severe pulmonary damage by activating the A2B receptor; whereas, treatment with its antagonist PSB1115 counters the effect of simvastatin. 135Another A2B agonist BAY 60−6583 is shown to decrease microvascular permeability and neutrophil migration into the pulmonary interstitium, 136 thus attenuating lung inflammation and edema in ALI mouse models. 137However, in the trauma-hemorrhagic shock-induced rat model, BAY 60−6583 can reduce pulmonary permeability but fails to alleviate neutrophil infiltration and inflammation in the lung. 138A newly found anti-inflammatory factor, netrin-1 dampens pulmonary inflammation and enhances alveolar fluid clearance to alleviate pulmonary edema in LPS-challenged ALI through the activation of the A2B receptor.The beneficial effect of netrin-1 is abolished by specific A2B receptor inhibitor PSB1115. 139Coherently, A2B receptor knockout mice show augmented mortality associated with excess inflammatory response upon LPS stimulation compared to wild-type controls. 140Furthermore, the activation of adenosine receptors either by adenosine or 5′-N-ethylcarbox-amidoadenosine restores vascular barrier functions and reduces inflammatory injury in LPSinduced ALI mice. 141These results support that adenosine receptor activation may offer a novel therapeutic approach for the management of ALI/ARDS (Figure 5C and Table 3).

Therapeutic agents targeting neuropeptides
VIP is a neuropeptide with multiple immunomodulatory effects.Lentivirus-mediated VIP addition has been reported to attenuate ALI in LPS-induced mouse models via inhibiting reactive oxygen species generation, NLRP3 inflammasome activation, and pro-inflammatory IL-17A expression in macrophages. 93,94Sensory C-type neurons-derived α-CGRP is one of the most abundant neuropeptides in the lung and possesses modulatory effects on immune functions.In an ALI rat model, LPS installation increases α-CGRP level but reduces α-CGRP receptor expression in the lung.Furthermore, exogenous α-CGRP improves oxygenation and lung injury-related index in ALI rats, which is associated with the upregulation of the transcription factor ICER (inducible cyclic adenosine monophosphate early repressor). 95Mechanistically, CGRP modulates macrophage polarization and inhibits inflammatory response in murine macrophages stimulated by LPS. 142Moreover, CGRP receptor antagonist CGRP8-37 exacerbates LPS-induced lung tissue damage in a rat model, accompanied by excessive proinflammatory cytokines expression, as well as decreased levels of aquaporin-1 and −5. 97Controversially, some scholars believe that CGRP is a mediator of neurogenic inflammatory response in several lung diseases caused by airway noxious stimuli.In an ovine ALI model of combined burn and smoke inhalation, pretreatment with a specific CGRP receptor antagonist BIBN4096BS attenuates early airway hyperemia, transvascular fluid flux and abnormalities in respiratory gas exchange. 96Accordingly, regulating pulmonary CGRP may become a potential therapeutic strategy for ALI/ARDS, but more studies are needed to clarify the concrete etiology when considering the neuropeptide.
Inhaled leytragin, an agonist of the δ-opioid receptor, is demonstrated to inhibit HMGB1 secretion in LPS-induced ALI of mice by preventing hyperacetylation at lysine residues and promoting sirtuin 1 to deacetylate HMGB1. 143inins are critical pro-inflammatory peptides and act on B1 and B2 receptors.Treatment with B1 receptor antagonist BI113823 significantly mitigates LPS-challenged direct lung injury and sepsis-induced pulmonary inflammatory response, as well as improves survival after severe polymicrobial sepsis. 144In an ALI mouse model, the bradykinin B1 receptor expression elevates upon LPS inhalation; whereas, posttreatment with its antagonist R-954 prevents cytokines/chemokines expression, leukocyte infiltration and protein leakage in the BALF, and decreases the airway hyperreactivity. 145Altogether, the data implicate that the kinin system, acting through the B1 receptor, participates in the pathogenesis and development of ALI (Figure 5D and Table 4).

Agents acting on the RAS signalling pathway
Studies have declared that RAS plays a vital and bidirectional role in multiple diseases.In the animal model, LPS or hyperoxia exposure increases the Ang II level and activates the RAS, leading to the occurrence and progression of ALI. 146,147Vitamin D exerts anti-inflammatory and anti-fibrotic effects in various pulmonary disorders.A vitamin D agonist, calcitriol, is proved to be beneficial against ALI by muffling LPS-induced lung permeability, which may be at least partially attributed to the expression balance of RAS members. 148In fact, subcutaneous Ang II infusion alone can establish the ALI mouse model by pathological identification; IL-22 administration shows protective effects on lung edema, inflammatory cell infiltration and vascular endothelial barrier damage in the mice by promoting nuclear translocation of STAT3. 149he biological activity of Ang II hinges on its interaction with distinct ATRs, and the pathological effect is mainly achieved by activating AT1R.Researchers have reported that endogenous Ang II aggravates pathogenetic conditions in ALI rats via AT1R, since AT1R antagonist losartan or ZD7155 prevents inflammatory NF-κB activation and pneumocytic apoptosis, and improves the alveolar fluid clearance. 146,150,151Fraxinol, a simple coumarin compound, can rebalance the expression between Ang II-AT1R and Ang (1-7)-Mas axis in the presence of LPS, thereby exerting anti-inflammatory and anti-apoptotic actions both in ALI mice and LPS-stimulated macrophages. 152omparatively, AT2R mediates the opposite effect of AT1R and is able to abrogate Ang II-AT1R axis-induced excessive inflammatory response and oxidative stress.Compound 21 is a highly selective AT2R agonist and shows superior efficacy both in pulmonary fibrosis and novel coronavirus pneumonia.Our most recent study verifies that Compound 21 alleviates acute inflammation and tissue damage in the lungs of LPS-challenged ALI mice via reprogramming macrophage function. 153AS activation can also protect against the development of ALI.Lipoxin A4, a product of arachidonic acid metabolism, has been found to protect ALI mice from lung damage via upregulating the ACE2-Ang (1-7)-Mas receptor axis in the lung. 154Besides, Ang (1-7) or its analogue AVE0991 attenuates the key features of ALI, such as neutrophil infiltration, lung edema and pulmonary vascular resistance caused by a ventilator, acid aspiration or oleic acid infusion. 155,156ACE2 is a critical negative regulator of RAS and exhibits beneficial properties on severe ALI caused by various etiologies. 157,158For instance, recombinant ACE2 remarkably reverses SARS-CoV-2 spike receptor-binding domain protein-induced ALI by directly cleaving AngI/AngII, further curbing NOX1/2 expression and their mediated inflammation and oxidative stress. 159ecombinant B38-CAP is a bacteria-derived ACE2-like carboxypeptidase and is more efficient than recombinant human ACE2.In the abdominal sepsis-or acid aspirationstimulated ALI model, B38-CAP degrades lung Ang II to Ang (1-7), leading to the downregulated cytokine generation, decreased inflammatory injury and improved survival rate of the mice. 160In addition, B38-CAP also shows beneficial efficacy in SARS-CoV-2-infected hamsters or human ACE2-transgenic mice. 161Treatment of ACE2 activator resorcinolnaphthalein or Ang (1-7) reduces the severity of LPS-induced ALI and pyroptosis, while AngII, ACE2 inhibitor MLN-4760 or Mas inhibitor A779 significantly exaggerates them, suggesting the protective role of ACE2/Ang (1-7)/Mas axis in the pyroptosis of ALI model. 162everal traditional Chinese medicines can also protect against ALI through RAS modulation.In an ALI rat model, paraquat-augmented airway inflammation, vascular leakage, and tissue damage are attenuated by tanshinone IIA (also known as danshen), an active compound isolated from Salvia miltiorrhizae Bunge, via the increased expression of ACE2 and Ang (1-7). 163Another example is Sini decoction, which has been reported to ameliorate sepsisor E. coli-induced lung injury in the mouse model via activating the ACE2-Ang (1-7)-Mas axis to exert the antiinflammatory effect. 164,165Osthole, a natural coumarin extracted from the fruit of Cnidium monnieri (L.) Cusson, is found to exhibit beneficial efficacy on LPS-challenged ALI mice.Pretreatment with osthole reduces inflammatory mediators secretion, pulmonary vascular leakage, as well as mortality in mice with severe lung damage.Furthermore, osthole markedly prevents the decreased expression of ACE2-Ang (1-7) in the lung of ALI mice, while ACE2 inhibitor blocks the protective effects, implying the potential role of RAS in the anti-inflammatory activity of osthole in ALI therapy. 166Collectively, the researchers provide evidence that drugs modulating RAS activation may become a conceptually new strategy for the clinical therapy of ALI with different etiologies, including the 2019 novel coronavirus (Figure 5E and Table 5).

CONCLUDING REMARKS
Building on a series of studies performed decades ago, the neuroimmune crosstalk has become a hot research topic in the field of both neurobiology and immunology.The positive feedback between the immune and neuronal systems in lung tissues may be an amplifying mechanism to induce potent inflammatory reactions, which appear to cause health problems, such as pulmonary infection, asthma, COPD and ALI/ARDS.Therefore, the modulation of neuroimmune interaction is a promising therapeutic target for these diseases.To expand the research field in the coming future, it is necessary to investigate the influence of the peripheral nervous system and its neurotransmitters or neuropeptides on the pulmonary immune microenvironment, as well as summarize potential drugs targeting neuroimmune crosstalk in lung inflammatory diseases represented by ALI/ARDS (Figure 6).Although these strategies have been widely accepted experimentally, very few have entered clinical trials to date, and none have been approved for clinical practice.Lessons have been drawn from these failures.First, the efficacy, stability, and safety of the therapeutic agents, and whether they would impair local or systemic immunity should be taken into account in future clinical development.More significantly, since danger signals or infection pathogens frequently activate multiple neuroimmune pathways, and single-targeted drugs failed in clinical trials; developing more potent remedies acting through multiple peripheral nervous system signallings, or combined with other anti-inflammatory therapeutics, may bring new hope for the treatment of ALI.Due to its heterogeneity, full consideration should be given to the specific type of ALI when applying a certain drug in order to avoid ineffective or even counterproductive effects.Besides, many ALI patients with underlying diseases already have an altered neuroimmune pattern, which may render previously effective drugs ineffective.For example, patients combined with COPD are likely to develop an enhanced cholinergic innervation, while patients suffering from chronic heart failure tend to have an overactivated RAS system.On this issue, choosing drugs with good targetability and minimal side effects or combined pharmacotherapy seems to be a better choice.
Furthermore, with the rapid progress of life sciences and biotechnology, new techniques could be used to seek a breakthrough.For instance, single-cell RNA sequencing can be applied to identify novel biomarkers and cell types, as well as explore the influence of neuroimmune crosstalk on cellular heterogeneity in ALI.CRISPR-CAS9 gene editing technology could be utilized to modify the expressions of neurotransmitters, neuropeptides and their receptors in specific cells, thus providing more precise tools to develop novel gene-editing drugs.Besides, due to the size advantage, nano-devices have better bio-distribution and could be functionalized to meet the desired pharmacokinetic and pharmacodynamic profiles.Therefore, nanomedicine and nano-delivery systems will shine the way to promote the clinical application of such a promising strategy in the near future.

A C K N O W L E D G E M E N T S
We would like to extend our heartfelt appreciation to Dr. Kun Wang and Dr. Wujian Xu for their valuable input and collaboration on this work.We also thank the anonymous reviewers for their constructive suggestions and comments, which greatly improved the quality of this manuscript.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.
Di Wu: Investigation and writing-original draft.Ximing Liao: Investigation and visualization.Jing Gao: Visualization.Yixuan Gao: Validation and writing-review & editing.Qiang Li: Conceptualization and funding acquisition.Wei Gao: Project administration; funding acquisition and writing-review & editing.
TA B L E 1

TA B L E 2
The development status of agents targeting sympathetic-immune pathway. 45 The development status of agents targeting neuropeptides.